| Objective The aim of this study is to compare the combination of osteopontin (OPN) and its receptor CD147via cyclophilin C(PPIC) in normal human knee synoviocytes and OA synoviocytes, then observe their combination impacting on the growth and proliferation of synoviocytes, and downstream cytokines(MMP-3and TNF-α), which could reveal the role and status of OPN and CD147in OA.Methods1. Cultured the normal human knee synoviocytes and OA synoviocytes in vitro.2. To construct the shRNA lentiviral expression vectors of SPP1and BSG. After generated by packaging of293T, the recombinant lentivirus were purified and transfected into normal human knee synoviocytes and OA synoviocytes.3. Quantitative reverse transcription polymerase chain reaction (qPCR) was used to assess the expression of OPN, CD147, PPIC, MMP-3and TNF-αin normal knee synoviocytes and OA synoviocytes. Bond of OPN and CD147via PPIC were detected with co-immunoprecipitation.4. Synovial cells were intervented as follow: N-A: normal synoviocytes; N-B:normal synoviocytes+OPN; N-C:normal synoviocytes+OPN shRNA; N-D: normal synoviocytes+CD147; N-E:normal synoviocytes+CD147shRNA; N-F:normal synoviocytes+OPN+CD147; N-G:normal synoviocytes+OPN+CD147+PPIC; OA-A:OA synoviocytes; OA-B: OA synoviocytes+OPN; OA-C:OA synoviocytes+OPN shRNA; OA-D: OA synoviocytes+CD147; OA-E:OA synoviocytes+CD147shRNA; OA-F:OA synoviocytes+OPN+CD147; OA-G:OA synoviocytes+OPN+CD147+PPIC. Cell colony formation was observd to evaluate the growth and proliferation by MTT after treatment. And Western blot was used to assess the expression of MMP-3and TNF-a in different treatments.Results1. Complex enzyme digestion using type II collagenase and trypsin was used to isolate and culture the primary synovial fibroblasts. The immunocytochemistry results were stable since the4th generation.3. OA synoviocytes grew faster than normal cells.2. ShRNA Lentiviral vectors of SPP1and BSG were successfully constructed and packaged as high-titer lentivirus particles. The virus titer of MOI100was high enough to ensure efficient transfection.3. The expression of OPN, CD147, PPIC, MMP-3and TNF-a mRNA in normal synoviocytes was1.00324022±0.076389036,0.99952589±0.045154528,1.00506933±0.110442982,1.00597389±0.10024154,1.00200722±0.077274527; The expression of OPN, CD147, PPIC, MMP-3and TNF-a mRNA in OA synoviocytes was3.62885022±0.350909902,3.45538689±0.307717070,11.46923478±0.199208822,3.27354856±0.303610376,4.59285122±0.374560116; The difference was significant between normal and OA synoviocytes. In normal synoviocytes, OPN cannot combine CD147without exogeneous PPIC. However, in OA synoviocytes, OPN can combine CD147in the case of with or without exogeneous PPIC.4. The growth rate of normal synoviocytes and OA synoviocytes were:59.67±2.52å'Œ61.67±3.21(P>0.05); The proliferation rate of normal synoviocytes and OA synoviocytes were:1.000±0.048å'Œ1.000±0.054(P>0.05). The clone formation assay and MTT results showed that in the same kind of synoviocytes:group A vs B, A vs D(P<0.05); group B vs C, D vs E(P<0.05); group F vs G, B vs G, D vs G(P<0.05); group B vs F, D vs F(P<0.05).5. The expressiong of MMP-3in group G and F of normal synoviocytes were0.341094182±0.017054709and0.211719491±0.010585975(P<0.05); The expressiong of MMP-3in group G and F of OA synoviocytes were0.780861988±0.039043099and0.540081853±0.027004093(P<0.05). The expressiong of TNF-ain group G and F of normal synoviocytes were0.61555±0.030777and0.501567±0.025078(P<0.05); The expressiong of TNF-ain group G and F of OA synoviocytes were1.276453±0.063823and1.023901±0.051195(P<0.05).Conclusion1. Compared with the normal synoviocytes, OA synoviocytes expressed more OPN, CD147, PPIC, MMP-3and TNF-α.2. Normal synoviocytes and OA synoviocytes were of similar rate of growth and proliferation.3. By combined with CD147in the process of osteoarthritis, OPN started downstream pathways, upgraded MMP-3and TNF-a expression, and accelerated cell growth and proliferation Thus simulating synovitis and synovial hyperplasia change. |
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